Glutathione depletion is the earliest detectable biochemical event in the Parkinsonian substantia nigra (SN),[unreadable] occurring prior to selective loss of mitochondrial complex I (CI) activity associated with the disease. We have[unreadable] Dreviously demonstrated that down-regulation of total glutathione (GSH + GSSG) levels in cultured[unreadable] dopaminergic cell lines results in decreased mitochondrial function linked to a selective decrease in CI[unreadable] activity. Loss of CI activity following acute glutathione depletion appears to be due to[unreadable] reversible nitrosylation of protein subunits comprising this complex. The effects of prolonged chronic[unreadable] glutathione depletion on CI activity in dopaminergic cells, however, are unknown but may involve[unreadable] additional, irreversible oxidative events. A major goal of our project is to assess CI inhibition in our[unreadable] dopaminergic cell model at various times and levels of glutathione depletion in order to identify the limits of[unreadable] function vs. dysfunction including the thresholds for reversible vs. irreversible inhibition as well as the[unreadable] oxidant species and protein targets involved. Once such targets have been identified, we will assess the[unreadable] presence of similar alterations in a newly constructed dox-inducible antiGSH transgenic mouse model[unreadable] following titration of glutathione levels within SN dopaminergic neurons in vivo as a model for molecular[unreadable] events associated with Parkinson's disease.[unreadable] Acute reduction in glutathione in our cell model also results in inhibition of the GSSG reducing enzyme[unreadable] glutathione reductase (GluRd) and up-regulation of the enzyme g-glutamyl transpeptidase (GGT) which[unreadable] breaks down extracellular GSH to substrates which are transported back into the cell for glutathione[unreadable] synthesis. The former may contribute to the detrimental effects of glutathione depletion on mitochondrial[unreadable] function while the latter appears to be a compensatory event. We will also explore the molecular[unreadable] mechanisms involved in these molecular alterations following titration of glutathione pools including the[unreadable] oxidants and the enzyme targets involved and if/how these changes modify mitochondrial function. We[unreadable] will assess the effects of both transgenic GSSG Rd and GGT expression on CI activity and mitochondrial[unreadable] function in our dopaminergic cell model to see if they act to attenuate any detrimental mitochondrial effects.[unreadable]